Rab3D: a regulator of exocytosis in non-neuronal cells.

Rab3D, a small Ras-like GTPase, is a key regulator of intracellular vesicle transport during exocytosis. It has been shown that Rab3 GTPases are abundant in cells with regulated secretory pathways and are thought to confer the specificity of docking and fusion during regulated exocytosis. Unlike other Rab3 isoforms, Rab3D is enriched in a number of non-neuronal tissues and is localised to secretory granules in the cytoplasm of these cells. The structure of Rab3D exhibits all of the conserved domains from the Rab family and also contains hypervariable N- and C-terminal regions. Rab3D undergoes post-translational isoprenylation and cycles between GDP- and GTP-bound forms. Apart from the factors involved in the Rab activation cycle, few Rab3D effector proteins have been identified to date. Nevertheless, it has long been suggested that Rab3D plays a role in regulated exocytotic processes as well as apically directed transcytosis. This review summarises the recent work on the biological function, structural integrity and molecular interactions of Rab3D in non-neuronal cells.

Prediction of maximal heart rate in individuals with mental retardation.

PURPOSE: It is well known that individuals with mental retardation (MR), especially those with Down syndrome (DS), have low maximal heart rates (MHR). We evaluated the ability to predict MHR in individuals with MR and DS in comparison with persons without MR. METHODS: Subjects completed a maximal exercise test on the treadmill with metabolic and HR measurements. Stepwise multiple regression was used to develop prediction equations for subjects with MR (N = 276; 97 with DS) and without (N = 296) MR, ranging in age from 9-46 yr. RESULTS: Subjects with MR exhibited significantly lower MHR (177 vs 185 beats.min(-1)) and VO2peak (33.8 vs 35.6 mL.kg-1.min(-1)). In subjects with MR, age was a poor predictor of MHR, Y = 189 - 0.59 (age) (R = 0.30, SEE = 13.8 beats.min-1; P < 0.01), but age was a better predictor for subjects without MR, Y = 205 - 0.64 (age) (R = 0.52, SEE = 9.9 beats.min(-1); P < 0.01). A large sample Z test indicated that these regression coefficients were significantly different (P < 0.01). However, adding DS to the regression improved the prediction for subjects with MR, Y = 210 - (0.56 age) - (15.5 DS) (R = 0.57; SEE = 11.8 beats.min(-1), P < 0.01). CONCLUSION: MHR can be predicted with similar accuracy in subjects with and without MR, provided DS is accounted for in the equation for the subjects with MR.

Do MSH6 mutations contribute to double primary cancers of the colorectum and endometrium?

Mismatch repair (MMR) gene mutations cause hereditary nonpolyposis colorectal cancer (HNPCC), a common form of familial colorectal cancer. Among MMR genes, germline MSH6 mutations are often observed in HNPCC-like families with an increased frequency of endometrial cancer. We have previously shown that a proportion of women affected with double primary cancers of the colorectum and endometrium carry germline MSH2 or MLH1 mutations and, thus, belong to HNPCC families. In this study, we have investigated the specific contribution of MSH6 defects to such double primary patients. By sequence analysis of the entire coding region of MSH6, three putative missense mutations were identified in patients with atypical family histories that do not meet HNPCC criteria. Moreover, one of these mutations, a novel substitution Arg901 His, was found in a patient previously shown to carry a truncating germline MLH1 mutation. Thus, MSH6 mutations are likely to contribute to the etiology of double primary cancers of the colorectum and endometrium.

Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by germline defects of mismatch repair (MMR) genes. Endometrial cancer is the most common extracolonic neoplasm in HNPCC and is the primary clinical manifestation of the syndrome in some families. The cumulative incidence of endometrial cancer among HNPCC mutation carriers is high, estimated to be from 22 to 43%. We hypothesized that women with double primary cancers of the colorectum and endometrium are likely to be members of HNPCC families. In order to determine how frequently HNPCC manifests in the context of double primary cancers, we examined alterations of two MMR genes, hMSH2 and hMLH1, in 40 unrelated women affected with double primary cancers. These cases were identified using hospital-based and population-based cancer registries in Ontario, Canada. MMR gene mutations were screened by single-strand conformation polymorphism analysis and confirmed by direct sequencing. Eighteen percent (seven of 40) were found to harbor mutations of one of the two MMR genes. Analysis of colorectal and/or endometrial tumors of mutation-negative probands found microsatellite instability in seven of 20 cases. Six of seven mutation-positive probands had strong family histories suggestive of HNPCC. First degree relatives of mutation-positive probands had a very high relative risk (RR) of colorectal cancer (RR = 8.1, CI 3. 5-15.9) and endometrial cancer (RR = 23.8, CI 6.4-61.0). The relative risk of mutation-negative cases was 2.8 (CI 1.7-4.5) for colorectal cancer and 5.4 (CI 2.0-11.7) for endometrial cancer. We recommend that all double primary patients with cancers at these sites should have a genetic evaluation, including molecular analysis for HNPCC where appropriate.

Rapid analysis of epitope-paratope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ionization mass spectrometry.

Progress in therapeutic or prophylactic immune intervention in HIV-1 infections may only come about with a detailed understanding at the molecular/atomic level of how antibodies neutralize (inactivate) virus infectivity. Currently information on the molecular aspects of antibody-virus interaction comes predominantly from X-ray crystallography, a process that is dependent on the production of suitable crystals. NMR can also be valuable but is complex and time consuming, while mass spectrometry has been limited to matrix-assisted laser-desorption ionization (MALDI) analysis of peptides eluted from the cognate antibody. Here, we have used electrospray ionization mass spectrometry (ESI-MS) to detect directly the interactions of a novel 17-amino-acid microantibody (MicroAb) that has HIV-1-inhibitory activity, and peptides representing the V3 regions of primary HIV-1 strains isolated from Brazil (clade B) and Africa (clade A). The MicroAb is based on the third complementarity-determining region of the heavy chain (CDR-H3) of a murine monoclonal IGGI (F58) specific for the V3 loop of the gp120 envelope glycoprotein of HIV-1. ESI-MS proved to be rapid (taking < 3 h for the entire analysis), sensitive (analytes at 2 mmol/ml), and accurate (RMM estimation to 0.01-0.1%). With it, we showed that the MicroAb forms complexes with the V3 peptides, implying that its antiviral activity is mediated by binding directly to the virus particle. In addition, through controlled protease digestion of the V3 peptides, we concluded that the CDR-H3 MicroAb bound to RKXXXIGPGR, a region similar to the epitope of the whole IgG as determined by ELISA. We believe that the approach exemplified here will be applicable generally to the identification of groups involved in receptor-ligand interactions.

The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.

Cardiorespiratory capacity of individuals with mental retardation including Down syndrome.

This study evaluated the cardiorespiratory capacity of persons with MR with and without Down syndrome. Analyses of individual data records of maximal exercise tests with metabolic analyses were conducted on tests of 111 subjects (31 men and 16 women with DS; 35 men and 29 women without DS) from six participating centers. All centers used a walking treadmill protocol previously shown to produce valid and reliable maximal tests with this population. Peak oxygen uptake and peak minute ventilation were higher in men than in women (P < 0.006), and in subjects without DS (P < 0.006). Peak heart rate was also higher in subjects without DS (P < 0.006). Peak respiratory exchange ratio (RER) was higher in subjects without DS (P < 0.006). Using peak RER as a covariate did not change the results. An analysis of peak minute ventilation, heart rate and VO2 of subjects with a peak RER above 1.1 revealed the same results. These data show that individuals with mental retardation have low levels of peak VO2, consistent with low levels of cardiovascular fitness. Individuals with Down syndrome have even lower levels of peak VO2 than their peers without Down syndrome, a finding that is possibly mitigated by the lower peak heart rates of the individuals with Down syndrome.

Effects of aerobic training in adolescents with Down syndrome.

The purpose of this study was to evaluate the effects that aerobic training has on adolescents and young adults with Down syndrome. Fourteen individuals with Down syndrome (mean age = 17.7 yr) participated in a 10-wk walking/jogging exercise training study. A pre- and post-training walking treadmill test was performed to determine the following parameters: peak oxygen uptake (VO2, absolute and relative), minute ventilation (VE, l.min-1), heart rate HR, b.min-1), RER (VCO2/VO2), and time and grade to exhaustion. Following the pre-training evaluations, subjects were assigned to a control group (N = 4) or an exercise group (N = 10). The exercise group underwent a 10-wk walk/jog training program at a frequency of 3 times per week, for a duration of 30 min, and at an intensity of approximately 65-75% peak HR. Following training, both control and experimental groups showed no changes in peak VO2 (absolute and relative), VE, HR, and RER. The exercise group, however, did demonstrate a significant improvement in peak exercise time (and grade). Although the training program did not produce improvements in aerobic capacity, it did produce gains in walking capacity. It was concluded that the adolescents and young adults may not be able to improve their aerobic capacity when performing a walk/jog training program.

Maximal exercise testing of mentally retarded adolescents and adults: reliability study.

Few data are available regarding maximal exercise testing of mentally retarded individuals. No data are available on the reliability of maximal exercise testing of mentally retarded individuals. The purpose of this study was to determine the reliability of graded exercise testing of mentally retarded adolescents and adults. The testing was conducted at two geographically different centers. At Center A, 14 mentally retarded adolescents (11 boys, three girls) with Down syndrome, who were educable or trainable, were recruited from a nonresidential school. The subjects completed two Balke-Ware treadmill protocols until exhaustion. The treadmill time and heart rate (HR) were recorded. The time between tests was approximately one week. At Center B, 21 mentally retarded adults (14 women, seven men means IQ = 56) were recruited from local workshops and group homes. These subjects completed a treadmill walking protocol, with metabolic measurements, until exhaustion. The time between tests varied from one to four months. At Center A, the subjects achieved a mean treadmill time of 8.72min on test one and 8.84min on test two (means HR = 174 and 175bpm, respectively). The reliability coefficient between the two tests was .94. At Center B, the subjects achieved a mean V0(2)max of 27.2mL.kg-1.min-1 on test one and 26.9mL.kg-1.min-1 on test two. The reliability coefficient was .93. These data show that maximal exercise testing is reliable for these populations of mentally retarded individuals, exhibiting similar values to their nonretarded peers.

Osteopenia: its relation to menstrual disorders in female athletes.

Loss of bone density (osteopenia) is a problem which is usually associated with postmenopausal women; however, it is a problem which has recently been identified in some female athletes. It is well documented that changes in the hormonal cycle are responsible for the changes in bone density, although the exact etiology remains to be identified. Amenorrhea occurs with some female distance runners and is linked to abnormal hormonal cycles, notably, decreased estrogen levels, similar to those experienced by postmenopausal women. Recent research has identified significant bone density losses in these amenorrheic female runners, and some have identified an associated increase in stress fractures and other injuries. Knowledge regarding the potential problems of amenorrhea and appropriate physical therapy are essential for those working with young female distance runners. J Orthop Sports Phys Ther 1990;11(8):351-354.